Parkinson's Disease (PD) is a devastating neurodegenerative disorder estimated to affect six million people world wide. Although PD is a complex disorder, it is characterized by debilitating motor symptoms that result from the depletion, beyond a critical threshold, of midbrain dopaminergic neurons (mDAs) in the substantia nigra. While the normal aging process leads to partial loss of these neurons, some individuals suffer genetic or environmental insults that accelerate the drop below this threshold. Accordingly, the number of mDAs one is born with could play a significant role in determining disease onset. Thus, understanding the molecular processes involved in mDA neurogenesis will critically impact models of PD susceptibility and therapeutics. Therefore, the goal of this proposal is to elucidate the mechanism for setting the neurogenetic interval of mDA progenitors. Strong evidence suggests that the transcription factor Lmx1b acts as a timer, analogous to an hourglass, such that progenitors exit the cell cycle when it is depleted. Importantly, we hypothesize that the duration of Lmx1b expression is controlled by an auto-regulatory negative feedback loop with microRNA-135a- 2. In order to test this "timer" model, we will use conditional mouse genetics to spatio-temporally manipulate gene expression and investigate mDA progenitor cell regulation during the normal window of neuron production. Specific Aim 1 involves removing Dicer, the enzyme necessary for processing all known microRNAs, with a Shh::Cre recombinase driver, which is active in the progenitors that give rise to all mDAs. Through this loss-of-function model, we will analyze the role of microRNAs in directing the number and cycling ability of mDA progenitors. Specific Aim 2 utilizes a complementary gain-of-function approach, in which candidate microRNA-135a-2 is prematurely over expressed using the same Shh::Cre driver. We will examine whether early elevation in microRNA levels is sufficient to repress Lmx1b and cause subsequent reduction in the progenitor pool. Specific Aim 3 directly investigates the ability of Lmx1b to initate expression of microRNA- 135a-2 in vitro, through luciferase assays, and in vivo, through a conditional mouse model. Collectively, the completion of these aims will increase our understanding of the molecular mechanisms that determine the total number of dopamine neurons a person is born with and will provide insights into an individual's susceptibility to PD. Moreover, this information might improve our ability to engineer dopamine producing neurons that could be surgically implanted into patient's brains for long-term treatment of PD. PUBLIC HEALTH RELEVANCE: The completion of the proposed research will increase our understanding of the molecular mechanisms that determine the total number of dopamine neurons a person is born with, and will provide insights into an individual's susceptibility to Parkinson's disease (PD). Moreover, this information might improve our ability to genetically screen for PD risk factors and to engineer dopamine producing neurons that could be surgically implanted into patient's brains for long-term treatment of PD.